Despite an extensive research directed at characterizing the cytoskeletal protein defects associated with aging and age-related neurodegeneration, the nature of the modifications responsible for protein deposition, insolubility, and proteolytic resistance remain elusive. Of particular relevance are neurofibrillary tangles in Alzheimer's disease, spheroids in amyotrophic lateral sclerosis, and Lewy bodies in Parkinson's disease. Although one or more factors appear to induce a transition between monomeric and non-covalent-associated forms of the constituent proteins, mainly neurofilaments (NF) or tau, the applicant postulates, and has preliminary evidence, that oxidative stress processes lead to an irreversible "cementing" of protein aggregates, at least in part, through covalent crosslinking. Modification involves a combination of direct oxidation of protein side chains and adduction of products of glycoxidation and lipid peroxidation that is expected to be manifested in overlapping but distinct patterns of markers for the three disease states. Thus, knowledge of individual disease variations in the structure and extent of these markers is hypothesized to help clarify the mechanisms of disease pathogenesis and provide clues for designing ameliorative agents/approaches. The proposal represents a multidisciplinary collaboration among three mid-career investigators with expertise in organic/peptide chemistry (Sayre), protein/analytical biochemistry (Anderson), and neuropathology/immunobiochemistry (Perry), with the goal of defining the structural basis of irreversible protein deposition. The aims are (i) structural elucidation of the crosslink and non-crosslink-carbonyl oxidative modifications in the protein deposits using novel mass-spectral biochemical analyses, (ii) development of antibodies to oxidation-specific NF and tau conformational epitopes and to structurally defined oxidative lesions, and (iii) correlative evaluation of spatio-temporal distribution of the oxidative-specific markers across different brain regions and their pathobiological significance.